Pre-stage perfusion and ultra-high seeding cell density in CHO fed-batch culture: a case study for process intensification guided by systems biotechnology

Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flow-filtration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultra-high seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 10(6) cells/mL in perfusion mode. Subsequently, production in the intensified fed-batch started immediately and the product titer was almost doubled (1.9-fold) in an equivalent runtime and with comparable product quality compared to low-seeded cultures. Driven by mechanistic modelling and next-generation sequencing (NGS) the process had been optimized by selecting the media composition in a way that minimized cellular adaptation between perfusion and production culture. As a main feature, lactate feeding was applied in the intensified approach to promote cell culture performance and process scalability was proven via transfer to pilot-scale i.e., 20 L pre-stage perfusion and 80 L production reactor. Moreover, an earlier shift from a growth associated to a production stage associated gene expression pattern was identified for uHSD cultures compared to the reference. Overall, we showed that the described intensification strategy yielded in a higher volumetric productivity and is applicable for existing or already approved molecules in common, commercial fed-batch facilities. This work provides an in-depth molecular understanding of cellular processes that are detrimental during process intensification. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00449-020-02337-1) contains supplementary material, which is available to authorized users.